Project Description Gut microbiota have been associated with many different disorders, including cardiovascular disease. One common mechanism involves the production, from dietary components, of metabolites that enter the circulation and affect physiologic functions such as inflammation. We propose to perform a comprehensive screen of gut microbiota-derived metabolites that contribute to cardio-metabolic disorders. Using a panel of genetically diverse inbred strains of mice, we will identify microbes and microbiota-derived metabolites that associate with atherosclerosis, followed by validation in human cohorts and mechanistic studies in germ-free mice. The work will be done in three laboratories with complimentary skills: A. Lusis (genetics), F. Rey (microbiology), and Z. Wang (metabolomics). All of the investigators have worked together for several years. The proposal represents an extension of a screen we previously performed using a panel of 100 inbred strains of mice for atherosclerosis (900 mice total). In that screen, we observed over a 200-fold range of lesion development. We now propose to analyze the microbiomes (Aim 1) and plasma metabolomes (Aim 2) of the mice and to relate these to atherosclerosis traits. We will then prioritize the significant associations by studying these in an atherosclerosis case-control human population (Aim 3). Finally, we will study the mechanisms by which the metabolites affect disease using germ-free mouse models (Aim 4). In preliminary studies, the levels of trimethylamine-N-oxide, another microbe-derived molecule shown to contribute to human atherosclerosis, were significantly correlated with lesion development. And, using a subset of the panel, we identified two microbes (A. muciniphila and R. intestinalis) associated with cardiometabolic traits and showed that these exhibited the predicted effects when used to colonize mice. These preliminary studies provide strong validation for the overall approach. We anticipate identifying several novel metabolites associated with atherosclerosis and related traits, and exploring the underlying mechanisms. This should pave the way for novel therapies that target the microbiome.